Synthesis and evaluation of the cytotoxicity of a series of 1,3,4-thiadiazole based compounds as anticancer agents.

Objective(s): Nowadays, cancer is an important public health problem in all countries. Limitations of current chemotherapy for neoplastic diseases such as severe adverse reactions and tumor resistance to the chemotherapeutic drugs have been led to a temptation for focusing on the discovery and development of new compounds with potential anticancer activity. Materials and Methods: A new series of 1,3,4-thiadiazole-derived compounds (3a-3l) were synthesized. N-(5-Mercapto-1,3,4-thiadiazol-2-yl)-2-(4-methoxyphenyl) acetamide (2) was prepared through direct amidation of 4-methoxyphenylacetic acid (2) with 5-amino-1,3,4-thiadiazole-2-thiol using EDC (N-Ethyl-N-dimethylaminopropyl carbodiimide) and HOBt (Hydroxybenzotriazole). Then, various derivatives of benzyl chloride containing electron withdrawing and electron donating moieties were reacted with compound 2 to prepare compounds 3a-3l. In vitro cytotoxicity assessment using MTT method was applied and results are presented as IC50. Results: All the synthesized compounds were characterized by 1H-NMR and IR spectroscopy. Some of the synthesized compounds were also characterized using MS spectroscopy. Related melting points were also recorded. According to the obtained data from MTT assay, all compounds (3a-3l) demonstrated a higher cytotoxic activity against MDA-MB-231 breast cancer cell line in comparison with other cell lines. Conclusion: It is notable that four synthesized compounds 3h (IC50= 11 ± 0.18 µM), 3j (IC50= 10 ± 0.39 µM), 3k (IC50= 11 ± 0.77 µM) and 3l (IC50= 8 ± 0.69 µM) exhibited higher cytotoxic activity against MDA-MB-231 cell line compared to imatinib (IC50= 20 ± 0.69 µM) as the reference drug.


Introduction
Cancer is a general name for a group of more than 100 diseases in which cells belonging to a part of the body, begin uncontrolled proliferation (1). Nowadays, cancer is an important public health problem in all countries (2,3). For decades, conventional chemotherapy has been the most common type of anticancer pharmacotherapy (4). Cancer chemotherapy has been one of the major advances in the field of medicine in the last few decades. However, drugs administered for chemotherapy have a narrow therapeutic index and therefore, there is a high incidence of unwanted side effects (5,6).

Chemistry
All chemicals including starter materials, reagents and solvents were bought from Merck and Sigma-Aldrich companies. Thin layer chromatography was done using TLC sheets from Merck Company. NMR spectra were obtained by Brucker 200 MHz and chemical shifts are expressed as δ (ppm) with tetramethylsilane (TMS) as internal standard. The IR spectra were obtained by Shimadzu 470 spectrophotometer (potassium bromide disks). Melting points were determined using electrothermal melting point analyzer apparatus and are uncorrected. The mass spectra were run on a Finigan TSQ-70 spectrometer (Finigan, USA) at 70 eV. All cancerous cell lines were purchased from Pasteur Institute, Tehran, Iran.
Then, 5-amino-1,3,4-thiadiazole-2-thiol (2) was added to the mixture and stirring was continued for 24 hr. Acetonitrile was removed under reduced pressure, ethylacetate/water was added and aqueous layer was removed. Organic phase was washed three times by sodium bicarbonate, diluted sulfuric acid and brine. Drying was done by anhydrous sodium sulfate and filtration was applied for the removal of sodium sulfate. Organic layer was evaporated using rotary evaporator apparatus and a yellowish powder was obtained for compound 3. Diethyl ether was used for washing the obtained powder (18).

General procedure of the synthesis of compounds 3a-3l
All intended 3a-3l derivatives were synthesized according to scheme 1. Equimolar quantities of compound 3 and potassium hydroxide were mixed and heated for 5 min and then appropriate benzyl chloride derivative was added. Reflux condition was applied for 24 hr and thin layer chromatography (TLC) was used for determining the reaction end. The reaction medium was cooled by crushed ice and cool water. A white precipitate was obtained for all compounds 3a-3l.

MTT assay
The derivatives of 1,3,4-thiadiazole (compounds 3a-3l) were assessed regarding their cytotoxic activity at 0.1-250 µg/ml in three human cancer cell lines; PC3 cell (prostate cancer), U87-C-531 (gliobalstoma) and MDA-MB-231 (breast cancer). Cells from different cell lines were seeded in 96-well plates at the density of 10×10 4 viable cells per well and incubated for 24 hr to allow cell attachment. Cells were then incubated for another 24 hr (depending on the cell cycle of each cell line) with various concentrations of compounds 3a-3l. Cells were then washed in PBS, and 20 μl of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) solution (5 mg/ml) was added to each well. An additional 4 hr incubation at 37°C was done, and then the medium was discarded. Dimethyl sulfoxide (60 μl) was added to each well, and the solution was vigorously mixed to dissolve the purple tetrazolium crystals. The absorbance of each well was measured by ELISA plate reader (Anthous 2020, Austria) at the test wavelength of 550 nm against the standard reference solution at 690 nm. The amount of purple formazan production is proportional to the number of viable cells (18).

Results
Compound 3 was used for preparing compounds 3a-3l, as an intermediate material. Compound 3 and compounds 3a-3l were synthesized at room temperature and under reflux condition, respectively. Compound 3 was prepared with high yield (84%) as a yellowish powder and an average yield (54-72%) was obtained for final derivatives in the form of a creamy to white powder. Melting point of intermediate and final compounds was reported. A range of 154-213C was recorded for final compounds. 1 H-NMR, IR and MS spectra for the intermediate and the final compounds were obtained.
The cytotoxicity of all synthesized compounds against three cancerous cell lines was evaluated by MTT assay (Table 1). Overall, all compounds 3a-3l afforded higher cytotoxic activity against MDA-MB-231, breast cancer cell line and lower cytotoxic activity against U87-C-531, glioblastoma cell line. Substitution of chlorine atom at position 3 (meta) of the phenyl ring resulted in a higher cytotoxic property than other positions of the phenyl ring. Compounds 3a-3c with chlorine moiety rendered better activity against MDA-MB-231 cell line in comparison with other cell lines. Replacement of chlorine moiety with nitro group decreased the cytotoxic effect of compounds 3d-3f against all three cell lines. Nitro group substituent can be more beneficial at positions 3 (meta) and 4 (para) of the phenyl ring compared to position 2 (ortho). Substitution of methoxy at position 4 in compound 3h (IC50= 11 ± 0.18 µM) led to a cytotoxic effect against MDA-MB-231 cell line compared to imatinib (IC50= 20 ± 0.82 µM). As with chlorine, a similar trend like was also observed for fluorine substituent. Position 3 (meta) of the phenyl ring was the best position for fluorine to render its cytotoxic activity in compounds 3i-3k. Compounds 3i-3k like other synthesized compounds in this series exhibited higher cytotoxic activity against MDA-MB-231 breast cancer cell line compared to cytotoxic effect against PC3 and U87-C-531 cell lines. Compound 3j (IC50= 10 ±0.39 µM) with ortho fluorine moiety and compound 3k (IC50= 11 ± 0.77 µM) with para fluorine moiety demonstrated higher cytotoxic activity against MDA-MB-231 cell line as compared with imatinib (IC50= 20 µM). Phenyl ring without any substituent (compound 3l) had the most beneficial cytotoxic effect (IC50= 8 ± 0.69 µM) in all synthesized compounds of this series.

Discussion
A new series of 1,3,4-thiadiazole based compounds was synthesized and their anticancer property was assessed by MTT assay, in vitro. Against PC3 cell line, Compound 3j with meta fluorine moiety showed the best cytotoxic effects. On the other hand, compound 3i with ortho fluorine substituent exerted the lowest anticancer activity in this series. This trend was also observed against U87-C-531 and MDA-MB-231 cell lines. U87-C-531 cell line was the most resistant cell line to the tested compounds in MTT assay and the recorded IC50 were not significant.
Overall, replacing electron withdrawing groups at position ortho of the phenyl ring was not capable of enhancing the cytotoxic activity. Fluorine moiety as an electron withdrawing group demonstrated a high cytotoxic potency when substituted at positions meta and para. Interestingly, compound 3l without any electron withdrawing and donating moiety on the phenyl ring, rendered the highest anticancer potency toward MDA-MB-231, PC3 and U87-C-531 cell lines (IC50= 8 ± 0.69 µM). Methoxy moiety as applied in compound 3h as an electron donating group caused an increase in the cytotoxic potency against MDA-MB-231 cell line when it was located at para position of the phenyl ring.

Conclusion
According to the obtained results, synthesized compounds could be proposed as potential anticancer lead compounds. The presented compounds exhibited robust anticancer activity against MDA-MB-231 (breast cancer) cell line, in vitro. Therefore, further investigation about the probable mechanism of these derivatives and also their in vivo activity evaluation is needed to be done in future studies. According to our data, ortho positioning of moieties was detrimental for cytotoxic activity. It suggests focusing on meta and para derivatives in the next explorations. Regarding the PC3 and U87-C-531 cell lines, more structural modification is necessary to enhance the cytotoxic potency. Overall, synthesis of new 1,3,4-thiadiazole derivatives can lead to the production of compounds with potential anticancer property, especially against breast cancer.